There is no rapid screening method available which reliably detects all point mutations in RNA molecules. The first part of this project is designed to improve current technologies in order to achieve this goal. Specifically, experiments will be designed to analyze RNA-RNA heteroduplexes in denaturing gradient gel electrophoresis and in solution in order to allow the rapid detection of mutations in mRNA molecules. In the second part of this grant, these techniques will be used to identify the mutations associated with Gaucher disease and phenylketonuria. The mutations will then be further characterized by directly sequencing the area of the RNA to which they have been localized. Subsequently, oligonucleotide probes will be synthesized and used to rapidly screen RNAs obtained from a large number of affected individuals, thus determining the frequency of each sequenced mutation in the affected population. Results obtained in these studies should contribute greatly to a better understanding of the genetic heterogeneity of genetic diseases and should also help us to correlate mutations with specific clinical disease patterns.